The present invention concerns an improved method for the analysis and detection of nicks in double stranded DNA.
The term xe2x80x9cnickxe2x80x9d is defined herein to mean a double stranded (ds) DNA fragment wherein one of the strands is contiguous and the complimentary strand contains at least one break, wherein two adjacent bases are not covalently linked. xe2x80x9cNicksxe2x80x9d in DNA fragments occur for a variety of reasons. For example, when long DNA fragments are constructed by enzymatic ligation of shorter strands, some fragments may not be completely ligated. Important and widely used products of DNA ligation comprise commercially produced DNA sequencing xe2x80x9claddersxe2x80x9d (BioRad, Inc., Richmond, Calif.; Life Technologies, Inc., Germantown, Md.). DNA xe2x80x9claddersxe2x80x9d are mixtures of DNA fragments, wherein the fragments comprise a defined range of base pair lengths and the fragments in the ladder differ by a constant base pair increment. For example, a 100 base pair (100 bp) ladder contains DNA fragments which differ by 100 bp increments over a range of 100 bp to 3,000 bp, i.e., 100 bp, 200 bp, 300 pb, 400 bp . . . 3,000 bp. Such DNA ladders are used as base pair length standards to calibrate electrophoresis gels. The accuracy of such ladders is of critical importance, since defective or impure ladders may lead to incorrect interpretation of sample results when compared to the standard base pair ladders. However, while a ladder containing nicks might be usable as a standard for gel electrophoresis, Applicants have found that such a ladder is not suitable for use as a standard using the more accurate and sensitive separation methods described herein.
Nicks are also formed when an enzyme which recognizes a base pair mismatch (mutation) in a heteroduplex, binds within the vicinity of a mutation and cleaves one strand of the DNA duplex which contains a non-complimentary base while leaving the other strand intact. Many such enzymes are known in the art and they are the basis of one form of mutation detection. For a comprehensive description of this subject see U.S. Pat. No. 5,763,178 to Chirikjian (1998). This reference and the references contained therein are incorporated in their entireties herein.
A nick in dsDNA cannot be detected by either gel or capillary electrophoresis of native DNA directly. Nicked strands can be detected, however, using denaturing polyacrylamide gels (Molecular Cloning, 2nd Ed. Sambrook et al. eds. Cold Spring Harbor Laboratory Press, 1989, incorporated herein by reference). Gel electrophoresis can also separate and detect nicked double stranded fragments which have been tagged with fluorescent or radioactive probes. However, this approach is costly and very labor intensive in that it requires the preparation of DNA fragments tagged with expensive probes. Methods which depend on gel electrophoresis (GEP) for separation of DNA fragments are subject to inherent deficiencies. This separation method is difficult to implement, not always reproducible, not accurate, difficult to quantify, and routinely takes five hours or more to complete (not counting set up time).
Other limitations in using GEP are related to the development and interpretation of bands on gels. The bands are often curved rather than straight, their mobility and shape can change across the width of the gel and lanes and bands can mix with each other. The sources of such inaccuracies stem from the lack of uniformity and homogeneity of the gel bed, electroendosmosis, thermal gradient and diffusion effects, as well as host of other factors. Inaccuracies of this sort are well known in the GEP art and can lead to serious distortions and inaccuracies in the display of the separation results. In addition, the band display data obtained from GEP separations is not quantitative or accurate because of the uncertainties related to the shape and integrity of the bands. True quantitation of linear band array displays produced by GEP separations cannot be achieved, even when the linear band arrays are scanned with a detector and the resulting data is integrated, because the linear band arrays are scanned only across the center of the bands. Since the detector only sees a small portion of any given band and the bands are not uniform, the results produced by the scanning method are not accurate and can even be misleading.
DNA molecules are polymers comprising sub-units called deoxynucleotides. The four deoxynucleotides found in DNA comprise a common cyclic sugar, deoxyribose, which is covalently bonded to any of the four bases, adenine (a purine), guanine (a purine), cytosine (a pyrimidine), and thymine (a pyrimidine), hereinbelow referred to as A, G, C, and T respectively. A phosphate group links a 3xe2x80x2-hydroxyl of one deoxynucleotide with the 5xe2x80x2-hydroxyl of another deoxynucleotide to form a polymeric chain. In double stranded DNA, two strands are held together in a helical structure by hydrogen bonds between, what are called, complimentary bases. The complimentarity of bases is determined by their chemical structures. In double stranded DNA, each A pairs with a T and each G pairs with a C, i.e., a purine pairs with a pyrimidine. Ideally, DNA is replicated in exact copies by DNA polymerases during cell division in the human body or in other living organisms. DNA strands can also be replicated in vitro by means of the Polymerase Chain Reaction (PCR).
Sometimes, exact replication fails and an incorrect base pairing occurs, which after further replication of the new strand results in double stranded DNA offspring containing a heritable difference in the base sequence from that of the parent. Such heritable changes in base pair sequence are called mutations.
In the present invention, double stranded DNA is referred to as a duplex. When the base sequence of one strand is entirely complimentary to the base sequence of the other strand, the duplex is called a homoduplex. When a duplex contains at least one base pair which is not complimentary, the duplex is called a heteroduplex. A heteroduplex duplex is formed during DNA replication when an error is made by a DNA polymerase enzyme and a non-complimentary base is added to a polynucleotide chain being replicated. Further replications of a heteroduplex will, ideally, produce homoduplexes which are heterozygous, i.e., these homoduplexes will have a complimentary, but altered sequence compared to the original parent DNA strand. When the parent DNA has the sequence which predominates in a natural population it is generally called the xe2x80x9cwild type.xe2x80x9d
Many different types of DNA mutations are known. Examples of DNA mutations include, but are not limited to, xe2x80x9cpoint mutationxe2x80x9d or xe2x80x9csingle base pair mutationsxe2x80x9d wherein an incorrect base pairing occurs. The most common point mutations comprise xe2x80x9ctransitionsxe2x80x9d wherein one purine or pyrimidine base is replaced for another and xe2x80x9ctransversionsxe2x80x9d wherein a purine is substituted for a pyrimidine (and visa versa). Point mutations also comprise mutations wherein a base is added or deleted from a DNA chain. Such xe2x80x9cinsertionsxe2x80x9d or xe2x80x9cdeletionsxe2x80x9d are also known as xe2x80x9cframeshift mutationsxe2x80x9d. Although they occur with less frequency than point mutations, larger mutations affecting multiple base pairs can also occur and may be important. A more detailed discussion of mutations can be found in U.S. Pat. No. 5,459,039 to Modrich (1995), and U.S. Patent No. 5,698,400 to Cotton (1997). These references and the references contained therein are incorporated in their entireties herein.
The sequence of base pairs in DNA codes for the production of proteins. In particular, a DNA sequence in the exon portion of a DNA chain codes for a corresponding amino acid sequence in a protein. Therefore, a mutation in a DNA sequence may result in an alteration in the amino acid sequence of a protein. Such an alteration in the amino acid sequence may be completely benign or may inactivate a protein or alter its function to be life threatening or fatal. On the other hand, mutations in an intron portion of a DNA chain would not be expected to have a biological effect since an intron section does not contain code for protein production. Nevertheless, mutation detection in an intron section may be important, for example, in a forensic investigation.
Detection of mutations is, therefore, of great interest and importance in diagnosing diseases, understanding the origins of disease and the development of potential treatments. Detection of mutations and identification of similarities or differences in DNA samples is also of critical importance in increasing the world food supply by developing diseases resistant and/or higher yielding crop strains, in forensic science, in the study of evolution and populations, and in scientific research in general (Guyer et al., Proc. Natl. Acad. Sci. USA 92:10841 (1995); Cotton, TIG 13:43 (1997)). These references and the references contained therein are incorporated in their entireties herein.
Alterations in a DNA sequence which are benign or have no negative consequences are sometimes called xe2x80x9cpolymorphismsxe2x80x9d. In the present invention, any alterations in the DNA sequence, whether they have negative consequences or not, are called xe2x80x9cmutationsxe2x80x9d. It is to be understood that the method of this invention has the capability to detect mutations regardless of biological effect or lack thereof. For the sake of simplicity, the term xe2x80x9cmutationxe2x80x9d will be used throughout to mean an alteration in the base sequence of a DNA strand compared to a reference strand. It is to be understood that in the context of this invention, the term xe2x80x9cmutationxe2x80x9d includes the term xe2x80x9cpolymorphismxe2x80x9d or any other similar or equivalent term of art.
Analysis of DNA samples has historically been done using gel electrophoresis. Capillary electrophoresis has been used to separate and analyze mixtures of DNA. However, in addition to the problems cited herein above, these methods cannot distinguish point mutations from homoduplexes having the same base pair length.
In addition to the deficiencies of denaturing gel methods mentioned above, these techniques are not always reproducible or accurate since the preparation of a gel and running an analysis is highly variable from one operator to another.
Recently, an HPLC method was introduced to effectively separate mixtures of double stranded polynucleotides, in general and DNA, in particular, wherein the separations are based on base pair length (U.S. Pat. No. 5,585,236 to Bonn (1996); Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993)). These references and the references contained therein are incorporated herein in their entireties. However, the reproducibility, accuracy, column life, and reliability of this method have not been adequately addressed. Aspects of DNA separation and mutation detection by HPLC which have been recognized and addressed by Applicants include the treatment of, and materials comprising chromatography system components, the treatment of, and materials comprising separation media, solvent pre-selection to minimize methods development time, optimum temperature pre-selection to effect partial denaturation of a heteroduplex during chromatography and optimization of the chromatographic separation for automated high throughput mutation detection screening assays. These factors are essential in order to achieve unambiguous, accurate and reproducible mutation detection results using HPLC. Applicants, through their own work, have gained an understanding of the unique mechanism involved in the liquid chromatographic separation of DNA and termed their separation method xe2x80x9cMatched Ion Polynucleotide Chromatographyxe2x80x9d (MIPC). This understanding has allowed Applicants to address the aspects related to the HPLC separation of polynucleotides in general and DNA in particular which have heretofore been unknown in the art.
There exists a need for a reliable method of calibrating a MIPC column to determine the relationship between the mobile phase composition and the base pair length of eluted fragments.
There exists a need for an accurate and reproducible analytical method for detecting nicked DNA which is easy to implement. Such a method, which in addition, can be automated and provide high throughput sample screening with a minimum of operator attention, is also highly desirable.
In one aspect, the present invention provides a method for determining the presence of xe2x80x9cnicksxe2x80x9d in a dsDNA fragment. In a further aspect, the invention provides an accurate method for determining the presence of a site of mismatch in a dsDNA sample. In another aspect the invention provides a method of calibrating a MIPC column by developing a relationship between the mobile phase composition and the retention time of dsDNA fragments having different base pair lengths, wherein the base pair lengths are within a specified base pair length range, such as a DNA ladder.
In still a further aspect, the invention provides an improved analytical method for the detection of mutations in nucleic acids. Yet another aspect is to provide a method for mutation detection which is highly reproducible, accurate, easily implemented and which can be automated for use with high throughput mutation detection assays or other analyses which require screening a large number of samples.
In one aspect, the invention provides a method for calibrating a Matched Ion Polynucleotide Chromatography column including the following steps: (a) applying to the column a sample containing a mixture of double stranded DNA fragments of known base pair length; (b) eluting the fragments; (c) detecting the eluted fragments; and (d) identifying the solvent concentration in the mobile phase at which each DNA fragment in the mixture elutes, whereby a relationship is derived between the organic solvent concentration in the mobile phase required for eluting DNA fragments of different base pair length from the column as a function of base pair length. In one embodiment of the invention, the DNA mixture is a DNA ladder. The DNA ladder can be pre-treated with a ligase to seal any nicks. In another embodiment, the mixture is a restriction enzyme digest.
In another aspect, the present invention is a method for determining the presence of a nick in a fragment of double stranded DNA by the following steps: (a) applying the fragment to a Matched Ion Polynucleotide Chromatography column; (b) eluting the fragment under fully denaturing conditions; (c) detecting the single stranded DNA species eluted in step (b); and (d) quantifying the single stranded DNA species from step (c) wherein at least three single stranded DNA species are detected if said fragment has a nick. The preferred denaturing conditions in step (b) are sufficient to completely denature the fragment. Examples of these conditions include an elevated level of at least one of the following: temperature (e.g. 60-80xc2x0 C.), urea concentration (e.g., about 5M), dimethylformamide concentration, organic solvent concentration, counterion concentration, and pH (e.g. pH 9-12).
In still another aspect, the invention is a method for determining the presence of nicked DNA in a sample of double stranded DNA fragments and includes the following steps: (a) applying a first aliquot of the sample to a Matched Ion Polynucleotide Chromatography column; (b) eluting the fragments in the first aliquot under non-denaturing conditions; (c) detecting the DNA species eluted in step (b); (d) determining the number of DNA species detected in step (c); (e) applying a second aliquot of the sample to a Matched Ion Polynucleotide Chromatography column; (f) eluting the fragments in the second aliquot under denaturing conditions; (g) detecting the DNA species eluted in step (f); (h) determining the number of DNA species detected in step (g); and (i) comparing the number in step (d) to the number in step (h) to determine whether or not the number in step (h) exceeds twice the number in step (d) whereby the presence of nicked DNA is indicated if the number in step (h) exceeds twice the number in step (d). The preferred denaturing conditions in step (f) are sufficient to completely denature the fragments. Examples of these conditions include an elevated level of at least one of the following: temperature (e.g. 60-80xc2x0 C.), urea concentration (e.g., 5M), dimethylformamide concentration, organic solvent concentration, counterion concentration, and pH (e.g. pH 9-12).
In yet another aspect, the invention provides a method for analyzing a sample of double stranded DNA to determine the presence of a mutation in the sample. The method includes: (a) contacting the sample with a mutation site binding reagent under conditions which allow the reagent to nick a strand of the double stranded DNA at or near the site of a mutation; (b) applying a first aliquot of the product of step (a) to a Matched Ion Polynucleotide Chromatography column; (c) eluting the fragments in the first aliquot under non-denaturing conditions; (d) detecting the DNA species eluted in step (c); (e) determining the number of DNA species detected in step (d); (f) applying a second aliquot of the product of step (a) to a Matched Ion Polynucleotide Chromatography column; (g) eluting the fragments in from step (f) under denaturing conditions; (h) detecting the DNA species eluted in step (g); (i) determining the number of DNA species detected in step (h); and (j) comparing the number in step (e) to the number in step (i) to determine whether or not the number in step (i) exceeds twice the number in step (e) whereby the presence of nicked DNA is indicated if the number in step (i) exceeds twice the number in step (e). In a preferred embodiment, the sample of double stranded DNA is the product of a hybridization of a DNA sample suspected of containing a mutation with corresponding wild type DNA. In a preferred embodiment, the mutation site binding reagent is an enzyme. Examples of suitable enzymes include S1 nuclease, mung bean endoucleose, CEL 1, mismatch repair enzymes, MutY protein, MutS protein, MutH protein, MutL protein, cleavase, exonuclease III, and HINF1. In another embodiment of this aspect of the invention, the mutation site binding reagent is a non-proteinaceous chemical reagent. A preferred chemical agent is an organometallic DNA intercalator. A preferred intercalator contains rhodium or ruthenium. Examples of suitable intercalators include bis(2,2xe2x80x2-bipyridyl)chrysenequinone diimine rhodium(III), bis(2,2xe2x80x2-bipyridyl)chrysenequinone diimine rhodium(III), (2,2xe2x80x2-bipyridyl)-bis(phenanthrenequinone) diimine rhodium(III), (bis(phenanthroline)dipyridophenazine ruthenium(II), and bis(phenanthroline)dipyridophenazine ruthenium(III).
In still another aspect, the invention provides a chromatographic method for analyzing a sample of double stranded DNA to determine the presence of a mutation in said sample, the method including the steps of: (a) separating a first aliquot of the sample using Matched Ion Polynucleotide Chromatography to produce a first chromatogram comprising peaks or other shapes (e.g. bands) which represent separated components of the sample; (b) contacting another aliquot of said sample with a mutation site binding reagent under conditions which allow the reagent to nick a strand of DNA at or near the site of a base pair mismatch; (c) separating the product of step (b) by the chromatographic method of step (a) to produce a second chromatogram; and (d) comparing the chromatogram of step (c) to the chromatogram of step (a), wherein a change in the retention time or the number of peaks or other shapes in the chromatogram of step (c) indicates the presence of a mutation in the original sample. In one embodiment, the separation of step (a) is performed under non-denaturing conditions. In a preferred embodiment, the separation of step (a) is performed under denaturing conditions. Also in a preferred embodiment, the sample of double stranded DNA is the product of a hybridization of a DNA sample suspected of containing a mutation with corresponding wild type DNA. In a preferred embodiment, the mutation site binding reagent is an enzyme. Examples of suitable enzymes include S1 nuclease, mung bean endoucleose, CEL 1, mismatch repair enzymes, MutY protein, MutS protein, MutH protein, MutL protein, cleavase, exonuclease III, and HINF1. In another embodiment of this aspect of the invention, the mutation site binding reagent is a non-proteinaceous chemical reagent. A preferred chemical agent is an organometallic DNA intercalator. A preferred intercalator contains rhodium or ruthenium. Examples of suitable intercalators include bis(2,2xe2x80x2-bipyridyl)chrysenequinone diimine rhodium(III), bis(2,2xe2x80x2-bipyridyl)chrysenequinone diimine rhodium(III), (2,2xe2x80x2-bipyridyl)-bis(phenanthrenequinone) diimine rhodium(III), (bis(phenanthroline)dipyridophenazine ruthenium(II), and bis(phenanthroline)dipyridophenazine ruthenium(III).